Endothelial dysfunction contributes to diabetic macrovascular complications, resulting in high mortality. Recent findings demonstrate a pathogenic role of P53 in endothelial dysfunction, encouraging the investigation of the effect of P53 inhibition on diabetic endothelial dysfunction. Thus, high glucose (HG)-treated endothelial cells (ECs) were subjected to pifithrin-alpha (PFT-alpha)-alpha specific inhibitor of P53, or P53-small interfering RNA (siRNA), both of which attenuated the HG-induced endothelial inflammation and oxidative stress. Moreover, inhibition of P53 by PFT-alpha or P53-siRNA prohibited P53 acetylation, decreased microRNA-34a (miR-34a) level, leading to a dramatic increase in sirtuin 1 (SIRT1) protein level. Interestingly, the miR-34a inhibitor (miR-34a-I) and PFT-alpha increased SIRT1 protein level and alleviated the HG-induced endothelial inflammation and oxidative stress to a similar extent; however, these effects of PFT-alpha were completely abrogated by the miR-34a mimic. In addition, SIRT1 inhibition by EX-527 or Sirt1-siRNA completely abolished miR-34a-Is protection against HG-induced endothelial inflammation and oxidative stress. Furthermore, in the aortas of streptozotocin-induced diabetic mice, both PFT-alpha and miR-34a-I rescued the inflammation, oxidative stress and endothelial dysfunction caused by hyperglycaemia. Hence, the present study has uncovered a P53/miR-34a/SIRT1 pathway that leads to endothelial dysfunction, suggesting that P53/miR-34a inhibition could be a viable strategy in the management of diabetic macrovascular diseases.